1. Field of the Invention
This invention pertains to T.sub.3 uptake test procedures and to a novel separating agent for use therein.
2. Description of the Prior Art
As early as 1939 (1), Treverrow reported that hormonal iodine compounds such as thyroxine constitute the major portion of the total serum iodine. Furthermore, these iodinated organic compounds could be distinguished from serum inorganic iodide because they are bound to serum protein. Since iodine constitutes 65 percent by weight of the thyroxine molecule, serum protein-bound iodine (PBI) was employed as an index of serum thyroxine (T.sub.4). This indirect measurement of serum thyroxine was believed to be a good indicator of the thyrometabolic status of an individual (2,3,4). The normal range of PBI values was found to be 4-8 ug/100 ml; values below 4 ug/100 ml were consistent with hypothyroidism whereas values above 8 ug/100 ml were indicative of thyrotoxicosis (hyperthyroidism).
A major pitfall of the PBI test is its inherent lack of specificity since high levels of inorganic iodide, radioopaque dyes and certain drugs give abnormally high values. In 1964, Murphy and Pattee (5) introduced their competitive protein binding analysis (CPB) for serum thyroxine which solved most of the nonspecificity problems associated with the PBI test. Due to the fact that CPB tests for serum thyroxine also required an extraction of the T.sub.4 from the remainder of the serum components, recovery variability has led to problems both in accuracy and precision.
Recently, radioimmunoassay (RIA) has become the method of choice for measuring serum thyroxine (6). The RIA technique can be run directly on serum without extraction and therefore yields a simple and yet highly specific test. In general; results from RIA are from 5 to 25 percent higher than those from CPB tests.
Although the direct measurement of serum thyroxine is not influenced by exogenous iodine, the value obtained will be influenced by the level of the circulating thyroxine binding proteins. A number of states which are totally unrelated to thyroid disease may cause abnormal serum levels of thyroxine. Changes in the serum level of circulating thyroxine binding proteins may cause the serum thyroxine level to be high or low even in the presence of normal thyroid function. Although the primary protein involved is thyroxine binding globulin (TBG), both thyroxine binding prealbumin (TBPA) and albumin also bind T.sub.4. Normally T.sub.4 is distributed as follows: 65% on TBG, 25% on TBPA, and 10% on albumin (7). In general, changes in TBG concentrations correlate much better with anomalies in thyroid function tests, such as the PBI or total thyroxine than do changes in TBPA (8).
Estrogen is one of the most important factors influencing the level of TBG, its effect being most notable in individuals receiving oral contraceptives or during pregnancy. In both cases TBG concentrations are markedly elevated and consequently due to the increased binding sites, the serum T.sub.4 or PBI are elevated above normal.
High serum thyroxine values are also found in euthyroid subjects who have increased levels of TBG (idiopathic increase of TBG) or have hyperprotenemia. Androgens have an opposite effect to estrogens in that they lower the TBG concentration and lead to falsely low serum T.sub.4 or PBI values. Other situations where the TBG concentration is below normal include an idiopathic decrease of TBG, the nephrotic syndrome and other hypoproteinimic states. Finally, a number of drugs such as diphenylhydantoin and salicylates, compete with thyroxine for binding sites on TBG, displace T.sub.4 from the TBG and thus result in falsely low serum values.
Although the most accurate method to measure TBG concentrations involves the electrophoretic method of Orsorio et al. (9), the technique is too cumbersome for routine use. The method of choice which has been used for this purpose is one of the many variations of the triiodothyronine (T.sub.3) uptake test. Hamolsky et al. (10) first performed this type of test and used red cells as the inert binder of T.sub.3. All T.sub.3 uptake tests are designed to assess the unsaturated binding capacity of serum proteins most notably TBG. The test is based on the fact that TBG binds T.sub.3 less firmly than T.sub.4 and therefore should not upset the equilibrium set-up between T.sub.4 and TBG and, further, T.sub.3 is not normally bound to TBPA.
In the T.sub.3 uptake test an equilibrium is developed between the patient's serum, added labeled T.sub.3 and an inert exogenous binder (separating agent) of the labeled T.sub.3. One must add a sufficient amount of labeled T.sub.3, e.g., .sup.125 I-labeled T.sub.3, to saturate the binding sites on the TBG after which the labeled T.sub.3 that is unbound is adsorbed by the separating agent and counted. Therefore, when the endogenous thyroxine level is increased, as in hyperthyroidism, serum TBG is relatively saturated and the T.sub.3 uptake will be high. Conversely in the hypothyroid state where thyroxine output is low, the labeled exogenous T.sub.3 will bind to the relatively unsaturated TBG yielding a low T.sub.3 uptake.
The major variations in T.sub.3 uptake methodology today are centered around the chemical nature of the separating agent. Ion exchange resins (11, 12, 13) hemoglobin saturated charcoal (14), Sephadex G-25 (15), inorganic crystalline materials such as magnesium silicate and aluminum silicate (16), and triiodothyronine antibody immobilized on the walls of polypropylene test tubes (17), all have been used as inert binders of labeled T.sub.3. Careful timing and controlled temperatures are necessary in the conventional T.sub.3 uptake tests using ion exchange resins, coated charcoal, or immobilized triidothyronine antibody as the separating agent. However, when the inorganic crystalline material of Eisentraut (17) was incorporated into this diagnostic test, the sorption of the free labeled T.sub.3 to this binder was neither critically time nor temperature dependent and this method gave highly efficient, fast and reproducible results.
Another T.sub.3 uptake assay employs an insolubilized colloidal suspension of bovine serum albumin as the separating agent to resolve the protein bound .sup.125 I T.sub.3 from the free .sup.125 I T.sub.3 (18).